The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. All references, including publications, patent applications, and patents, cited herein are incorporated by reference in full to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The analysis of biological analytes is critical for human health, safety and the environment. For example, infectious diseases can be diagnosed and treated by identifying the specific causes of the disease. This can be done by analyzing bodily samples using biological assays for the presence of disease-causing biological analytes including cells such as bacteria, protozoa and fungi, virus particles, toxins caused by the infectious materials, as well as biomolecular constituents of the infectious materials such as DNA, RNA and proteins.
Diseases, cancers and medical conditions such as cardiac arrest can be identified by the presence and levels of protein antigens and antibodies produced by the human immune system or other bodily mechanism. Genetic markers can also be used to indicate an abnormal state or predisposition to diseases, cancers and medical conditions. Hazardous biological materials can also be transmitted by infected food, plants, water, air, objects such as surfaces or containers, insects, birds, fish, lizards, rodents, animals, and people. Samples can be analyzed for pathogenic cells, virus particles, protein toxins, and biomolecules such as nucleic acids and proteins. Some hazardous biological materials are naturally occurring while others can be intentionally released by bioterrorists. Many other applications and sectors such as biotechnology, pharmaceutical, and forensic also require analysis for the identification, presence and levels or concentrations of biological analytes.
Accurate, timely and practical analysis of biological analytes is extremely complex. Some analytes can be present as substances that are difficult and costly to accurately assay. Some analytes are not specific to a single disease, cancer, or medical condition, and some diseases, cancers and medical conditions are not specific to a single analyte. Therefore identification of analytes can require multiplex assays for multiple analytes and in some cases multiple types of analytes for confirmation.
Some analytes can be present in extremely low levels and may not be detected by an assay, resulting in false negative outcomes. This requires highly sensitive assays and preferably the additional use of an amplification or enrichment process to increase the level of analytes before assaying.
Some analytes can be surrounded by non-specific materials in several orders of magnitude greater levels, as well as non-specific materials comprising non-specific strains and species of the target analyte which are physically and chemically similar. Non-specific materials can prevent the analytes from being detected by an assay, and result in false negative outcomes. In the case where the analyte is not present in the sample, the non-specific material may be incorrectly detected by the assay, causing a false positive outcome. This requires highly specific assays and preferably the additional use of a purification process to remove non-specific materials before assaying.
Even though some analytes may be present in a sample and correctly detected by the assay, some analytes can have an abnormal or harmful level which is higher or lower than a normal level. Some analytes have levels that change over time. This requires assays that can quantify analyte levels or concentrations, accurately and frequently.
Some analytes are highly infectious, extremely harmful, and costly to treat or remediate. These analytes need to be analyzed in a very timely manner to minimize the transmission of the infection. As well, some analytes have an elevated level for a limited period of time. Some assay operators have limited technical proficiency and need assays that are automated and easy to use. Some testing organizations have budgetary constraints and require assays to be low cost for consumables, labor, sample collection, assay equipment and laboratory facilities.
Numerous assays are known for detecting biological analytes in a sample. Four general types of biological analytes are cells, nucleic acids, proteins and redox active species. The technologies and assays directed at detecting these analytes are basically separate and independent. In certain cases different technologies can be used to measure the presence of analytes associated with the same disease. As an example, Table 1 illustrates the relative limits of detection and turnaround times for selected commercial products that use cell cultures, nucleic acid amplification tests and protein immunoassays for detecting analytes associated with certain infectious diseases. Cell cultures and nucleic acid amplification tests have the lowest limits of detection but also have longer turnaround times because of the test complexity, labor-intensity, and laboratory logistics. Protein immunoassays can be done in laboratories, and are also available as simple rapid point-of-care tests that have a higher limit of detection.
TABLE 1Relative Limits of Detection and Turnaround Times ofDifferent Detection Technologies Used by Commercial ProductsProteinProteinNucleic AcidImmuno-Immuno-Cell AmplificationassayassayAnalyteCultureTest(Lab Test)(POC Test)Limit of DetectionC. difficile1 pg/mL10 pg/mL300 pg/mL1000 pg/mLToxin ProteinCampylobacter3 × 1023 × 1033 × 1063 × 107C. jejunicfu/mLcfu/mLcfu/mLcfu/mLBacteriaHIV VirusNot~15~3000>>3000applicablevirions/mLvirions/mLvirions/mLto virusesTurnaround TimeTime between 2-7 days1-2 days1-2 days5-60 minsample and test result
Cell assays employ viable cells to reproduce outside of their natural environment to amplify the detection signals. Targets cells reproduce in a growth media incubated at an appropriate temperature, gas mixture and pH. Materials can be included to suppress the growth of non-specific cells. Detectable dyes provide color which intensifies with an increasing number of cells. Cell cultures are sensitive assays, but have a slow turnaround time (2-7 days) for producing a detectable number of cell colonies, and can result in false positive results caused by non-specific strains of the target cells that reproduce in the growth media. Cell assays can fail if target cells are unable to reproduce due to cells being dead or injured, or from contamination of the growth media. Because of the labor-intensive processing, cell assays can also fail from technician error due to an incorrect manual process, or from an inability to distinguish target cells from non-specific materials.
Nucleic acid assays cause a target region of DNA strands to amplify using polymerase chain reaction (PCR) during repeated thermally-induced biochemical processes. DNA fragments are exposed to appropriate denaturing conditions including high temperature to melt double helix DNA into single DNA strands. The temperature is lowered and target regions of the single stands act as templates which anneal with complementary nucleotide primers. The temperature is raised to an activity temperature where a polymerase enzyme causes a chemical reaction to synthesize new single DNA strands complementary to the single strand DNA templates, which form double helix DNA. The process is repeated until a sufficient number of copies are produced. Fluorescent dyes or fluorophore-containing DNA probes create a detectable signal which intensifies with an increasing number of target DNA fragments. Nucleic acid assays are highly specific and increase in sensitivity when more detectable target DNA fragments are produced. Because of the complex processes for sample preparation, amplification, detection and quantification, nucleic acid assays require highly skilled operators using costly equipment and expensive laboratory facilities. This limits the number of organizations that can conduct nucleic acid assays. Bottlenecks can occur at test labs and cause delays in testing, treatment and remediation. Nucleic acid assays can fail when non-specific DNA products amplify due to contamination or improper sample processing in advance of PCR. Failure can also occur if detectable fluorescent dyes or fluorophores are not adequately delivered along with the replicated target DNA fragments.
Protein assays identify and quantify proteins such as hormones and enzymes, by acting as antigens or antibodies in a chemical reaction. One of the most common protein assays is enzyme-linked immunosorbent assay (ELISA). In a direct ELISA an antigen analyte is adsorbed to a plate and a blocking agent is added to block potential binding sites from non-specific materials. An antibody-enzyme complex is added to bind with the antigen analyte and the plate is washed to remove unbound antibody-enzyme complexes. An appropriate enzyme substrate is added to produce an optical signal proportional to the amount of antigen analyte in the sample. In a Sandwich ELISA, a matched pair of antibodies forms a sandwich structure containing a first outer antibody layer to capture the analyte, an internal layer comprising the antigen analyte and a second outer antibody layer to detect the analyte. The capture antibody is initially bound to the plate and then binds with the antigen analyte contained in a test sample. After washing, a detection antibody-enzyme complex is added to bind with the antigen analyte and the plate is washed to remove unbound capture antibody-enzyme complexes. An appropriate enzyme substrate is added to produce an optical signal proportional to the amount of antigen analyte in the sample. Direct ELISA is faster because only one antibody is being used and fewer steps are required. Sandwich ELISA can have a lower detection limit because each capture antibody can contain several epitopes that can be bound by detection antibodies. Sandwich ELISA can also be made more sensitive using avidin-biotin complexes which have several sites for enzymes to provide multiple enzymes per analyte. This can amplification the detection signal by ten to a few hundred times. In contrast, cell cultures and PCR can produce millions or more copies. Protein assays are relatively easy to use, rapid and low cost. A major disadvantage is the inability to significantly amplify protein signals, making it necessary for the subject or its immune system to produce a detectable level of target protein analytes. This waiting period can delay detection and subsequent treatment by weeks or months. If the protein analytes are assayed using immunoassay before a detectable level is secreted, then a false negative detection outcomes will be produced causing the disease to be undetected.
Another problem is the specificity of antibodies and antigens. Many antibodies, and particularly polyclonal can detect a wide range of species; however these can include non-specific strains that produce false positive detection outcomes. The use of highly specific monoclonal antibodies greatly improves the specificity.
All of the abovementioned assays suffer from limitations. None of these assays can identify all types of analytes. Unlike cell and nucleic acid assays, protein assays cannot support significant signal amplification which can limit the sensitivity of protein assays. Amplification used in nucleic acid amplification tests and cell cultures adds time, cost and complexity. Cell and protein assays can have insufficient specificity and can benefit from purification steps such as magnetic separation. This adds to the assay cost and complexity. Quantification can be difficult if done manually or expensive if a transduction system is needed to convert optical signals to electrical signals. Nucleic acid amplification assays are sensitive and specific, however the complex processes used for sample preparation, amplification, detection and quantification require highly skilled operators, costly equipment, expensive laboratory facilities, and time-consuming laboratory logistics. This complexity limits the number of organizations that can conduct nucleic acid assays.
Another general type of biological assay is for redox species and works when a redox analyte electrochemically reduces and/or oxidizes at an electrode. A redox analyte is placed in close proximity to a set of electrodes and undergoes electrical stimulation such as applying a potential. This causes the analyte to lose electrons through oxidation or gain electrons through reduction, which can be measured as an electrical signal at the working electrode. The amount of analyte oxidized or reduced and the corresponding electrical signal reflect the quantity of analyte in the sample. Other materials may be also be present such as a mediator to transport redox electrons, and non-specific materials, both of which can cause electrical noise that interferes with the electrical signal from the analyte. When redox analytes are present in high levels, such as approximately 1014 glucose molecules in blood associated with 1.1 mmol/L, redox signals are relatively high compared with background noise and can be directly measured to provide rapid quantification with acceptable sensitivity and specificity. Since the detection signal is electrical, no expensive transduction system is needed to convert optical signals. This allows glucose meters using redox assays to be performed in rapid, easy to use, low cost instruments.
Other redox analytes can be present in very low levels such as approximately 104 to 106 guanine molecules associated with 5,000 copies/mL of HIV RNA in blood as required for clinical use. Low levels of guanine bases in nucleic acids such as RNA can be oxidized to generate very low electrical current signals while significant background noise currents are produced due to the relatively high potentials required for guanine oxidation. This makes it difficult to distinguish oxidation signals from background noise signals.
TABLE 2Examples of Redox AnalytesRedox Analytes Available forRedoxLevel Required forElectrochemicalAnalyteSampleClinical UseQuantificationGlucose1 μL whole1.1 mmol/L glucose~1014 glucoseblood(20 mg/dL)moleculesHIV100 μL whole5,000 RNA~104-106 guaninebloodcopies/mLmolecules
Various approaches have been employed to quantify nucleic acid analytes using redox assays by improving the signal-to-noise ratio. One approach reduces the active surface area of a biosensor working electrode by replacing a conventional solid working electrode with a nanobiosensor comprising randomly distributed forests of nanoscale structures on the electrode surface (Lieber, et al, Thorpe, et al). Another nanobiosensor approach replaces the randomly distributed forests of nanoscale structures with ordered arrays of nanoscale structures spaced at least 1 μm apart to further reduce the surface area of a working electrode (Gordon, et al). These approaches allowed the guanine signal to be better distinguished from noise over conventional solid working electrodes but not to the degree required for direct measurement of the low level of redox species associated with target bio-analytes such as guanine molecules. Fabrication of nanoscale structures, such as 100 nm diameter carbon nanotubes, provides additional complexity over microscale structures that result in the need for specialized production equipment with high cost and limited throughput, poor production yields, and high unit costs for nanobiosensors.
Another approach employs PCR to amplify target DNA before detection by a conventional biosensor (Ozkan, et al). The use of PCR provides added complexity, time and cost which negates the benefits experienced from the glucose redox assay.
Another approach employs magnetic separation to purify analytes by removing background interferences before detection by a conventional biosensor. Palesecek et al, and Wang and Kawde capture target sequences using probe DNA immobilized onto magnetic particles. After target hybridization, the particles are magnetically separated from the pool of analytes. The collected DNA is denatured in acidic solutions, and the free guanine and adenine nucleotides are collected and analyzed using anodic stripping voltammetry. Although the noise from other interferents can be reduced, the inherent background signal from water electrolysis always presents. As a result, the guanine oxidation signal is too low for direct measurement in the presence of such large background currents.
There is a need for an assay that can determine the presence and quantity of very low level analytes including multiple analytes and multiple types of analytes in the same sample, provide high sensitivity preferably with signal amplification, provide high specificity preferably with purification, and provide the above in a rapid, easy to use and low cost device, including the capability for point-of-care use.